Oxalic acid (oxalate) is a diffusable toxin associated with various plant diseases, particularly those caused by fungi. Some leafy green vegetables, including spinach and rhubarb, produce oxalate as a nutritional stress factor. When plants containing oxalate are consumed in large amounts, they can be toxic to humans.
Oxalate is used by pathogens to gain access into and subsequently throughout an infected plant. See for example, Mehta and Datta, The Journal of Biological Chemistry, 266:23548-23553, 1991; and published PCT Application WO92/14824.
Field crops such as sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut, clover, as well as numerous vegetable crops, flowers, and trees are susceptible to oxalate-secreting pathogens. For example, fungal species including Sclerotinia and Sclerotium use oxalic acid to provide an opportunistic route of entry into plants, causing serious damage to crops such as sunflower.
Because of the role of oxalate in plant disease and toxicity, compounds that inhibit oxalate mediated disease, and particularly genes encoding such inhibitory degrading molecules, are greatly needed.
Enzymes that utilize oxalate as a substrate have been identified. These include oxalate oxidase and oxalate decarboxylase. Oxalate oxidase catalyzes the conversion of oxalate to CO.sub.2 and H.sub.2 O.sub.2. A gene encoding barley oxalate oxidase has been cloned from a barley root cDNA library and sequenced (See PCT publication No. WO92/14824). A gene encoding wheat oxalate oxidase activity (Germin) has been isolated and sequenced, (PCT publication No. WO 94/13790) and the gene has been introduced into a canola variety. Canola plants harboring the gene appeared to show some resistance to Sclerotinia sclerotiorum, in vitro (Dumas, et al., 1994, Abstracts: 4th Int'l. Congress of Plant Molecular Biology, #1906).
Oxalate decarboxylase converts oxalate to CO.sub.2 and formic acid. A gene encoding oxalate decarboxylase has been isolated from Collybia velutipes (now termed Flammulina velutipes) and the cDNA clone has been sequenced (WO94/12622, published Jun. 9, 1994). Oxalate decarboxylase activities have also been described in Aspergillus niger and Aspergillus phoenices (Emiliani et al., 1964, ARCH. Biochem. Biophys. 105:488-493), however the amino acid sequence and nucleic acid sequence encoding these enzyme activities have not been isolated or characterized.
Enzymatic assays for clinical analysis of urinary oxalate provide significant advantages in sensitivity and qualification Obzansky, et al., 1983, Clinical Chem. 29:1815-1819. For many reasons, including reactivity with interfering analytes and the high cost of available oxalate oxidase used in this diagnostic assay, alternative enzymes are needed. (Lathika et al., 1995, Analytical Letters 28: 425-442).
In this application, we disclose the isolation, cloning, and sequencing of a unique gene encoding an oxalate decarboxylase enzyme from Aspergillus phoenices. The gene is useful in producing highly purified Aspergillus phoenices oxalate decarboxylase enzyme, in producing transonic plant cells and plants expressing the enzyme in vivo, and in diagnostic assays of oxalate.